Fenglan Jia

Interaction of Virion Protein Vpr of Human Immunodeficiency Virus Type 1 with Cellular Transcription Factor Sp1 and trans-Activation of Viral Long Terminal Repeat

Acquired immunodeficiency syndrome (AIDS) is a result of replication of the human immunodeficiency virus type 1 (HIV-1) predominantly in CD4 T lymphocytes and macrophages. However, most of these cells in vivo are immunologically quiescent, a condition restricting HIV-1 replication. Vpr is an HIV-1 virion protein suspected to enhance HIV-1 replication in vivo. We demonstrate in this report that Vpr specifically activates HIV-1 long terminal repeat (LTR)-directed transcription. This effect is most pronounced on a minimal promoter from HIV-1 LTR containing the TATA box and binding motifs for the ubiquitous cellular transcription factor Sp1. Evidence is presented that Vpr interacts with Sp1 when Sp1 is bound to the Sp1 motifs within the HIV-1 LTR. Both Vpr-Sp1 interaction and Vpr trans-activation require a central Leu/Ile-rich domain in Vpr. Our findings suggest that Vpr trans-activation through Sp1 is most critical for the immediate early transcription of HIV-1 when other positive regulators, such as NF-κB, are limited or inactive, a condition presumably present in vivo. By interacting with Sp1, Vpr also has the potential to influence cellular gene expression and cellular functions. Thus, therapeutic approaches directed toward blocking the Vpr trans-activation function could prove valuable in treating AIDS.

Neuropathogenesis of Chimeric Simian/Human Immunodeficiency Virus infection In Pig-tailed and Rhesus Macaques

We recently reported that a chimeric simian/human immunodeficiency virus (SHIVKU‐1) developed in our laboratory caused progressive depletion of CD4+T lymphocytes and AIDS within 6 months of inoculation into pig‐tailed macaques (M.nemestrina). None of the pig‐tailed macaques showed productive SHIV infection in the central nervous system (CNS). In this report, we show that by further passage of the pathogenic virus in rhesus macaques [M. mulatta], we have derived a new strain of SHIV (SHIVKU‐2) that has caused AIDS and productive CNS infection in 3 of 5 rhesus macaques infected with the virus. Productive replication of SHIV in the CNS was clearly shown by high infectivity titers and p27 protein levels in brain homogenates, and in 2 of the 3 rhesus macaques this was associated with disseminated, nodular, demyelinating lesions, including focal multinucleated giant cell reaction, largely confined to the white matter. These findings were reminiscent of HIV‐1 associated neurological disease, and our immunohistochemical and in situ hybridization data indicated that the neuropathological lesions were associated with the presence of SHIV‐specific viral antigens and nucleic acid respectively. However, the concomitant reactivation of opportunistic infections in these macaques suggested that such pathogens may have influenced the replication of SHIV in the CNS, or modified the neuropathological sequelae of SHIV infection in the rhesus species, but not in pig‐tailed macaques. Our findings in the two species of macaques highlight the complexities of lentiviral neuropathogenesis, the precise mechanisms of which are still elusive.

Chimeric SHIV that causes CD4+ T cell loss and AIDS in rhesus macaques

Abstract: By animal to animal passage in rhesus and pig‐tailed macaques, we developed a rhesus model of HIV‐1 disease in humans. Rhesus macaques infected with a cell‐free stock of SHIVKU‐2 developed CD4+ T cell loss, primary lentiviral encephalitis and pneumonia, and AIDS. Six of nine rhesus macaques died within eight months post‐inoculation, while the remaining three are at five, five, and eight months post‐inoculation, respectively. Animals infected by either mucosal or parenteral routes of infection had a similar course of infection.

A Noninfectious Simian/Human Immunodeficiency Virus DNA Vaccine That Protects Macaques against AIDS

ABSTRACT Simian/human immunodeficiency virus SHIVKU2 replicates with extremely high titers in macaques. In order to determine whether the DNA of the viral genome could be used as a vaccine if the DNA were rendered noninfectious, we deleted the reverse transcriptase gene from SHIVKU2 and inserted this DNA (ΔrtSHIVKU2) into a plasmid that was then used to test gene expression and immunogenicity. Transfection of Jurkat and human embryonic kidney epithelial (HEK 293) cells with the DNA resulted in production of all of the major viral proteins and their precursors and transient export of a large quantity of the Gag p27 into the supernatant fluid. As expected, no infectious virus was produced in these cultures. Four macaques were injected intradermally with 2 mg of the DNA at 0, 8, and 18 weeks. The animals developed neutralizing antibodies and low enzyme-linked immunospot assay (E-SPOT) titers against SHIVKU2. These four animals and two unvaccinated control animals were then challenged with heterologous SHIV89.6P administered into their rectums. The two control animals developed viral RNA titers exceeding 106 copies/ml of plasma, and these titers were accompanied by the loss of CD4+ T cells by 2 weeks after challenge. The two control animals died at weeks 8 and 16, respectively. All four of the immunized animals became infected with the challenge virus but developed lower titers of viral RNA in plasma than the control animals, and the titers decreased over time in three of the four macaques. The fourth animal remained viremic and died at week 47. Whereas the control animals failed to develop E-SPOT responses, all four of the immunized animals developed anamnestic E-SPOT responses after challenge. The animal that died developed the highest E-SPOT response and was the only one that produced neutralizing antibodies against the challenge virus. These results established that noninfectious DNA of pathogenic SHIV could be used as a vaccine to prevent AIDS, even though the immunological assays used did not predict the manner in which the challenge virus would replicate in the vaccinated animals.

Passively Administered Neutralizing Serum that Protected Macaques against Infection with Parenterally Inoculated Pathogenic Simian-Human Immunodeficiency Virus Failed to Protect against Mucosally Inoculated Virus

Macaques inoculated orally, vaginally, or parenterally with SHIV(KU-1) develop severe systemic infection, acute loss of CD4+ T cells, and AIDS. We showed in a previous report that passive immunization with neutralizing serum protected macaques against infection with parenterally inoculated pathogenic SHIV given 24 hr later. In the study reported here we asked whether the identical passive immunization protocol would protect macaques against infection with pathogenic SHIV following oral inoculation of the virus. Ten pigtail macaques were inoculated orally with one animal infectious dose of SHIV(KU-1). Four of the 10 had been given pooled anti-SHIV plasma (15 ml/kg) 24 hr earlier, 4 others were given the same dose of anti-SHIV plasma 2 hr after virus challenge, and the 2 remaining animals were used as controls. The neutralizing antibodies failed to protect macaques against infection after mucosal challenge with SHIV(KU-1).

Protection Against Late-Onset AIDS in Macaques Prophylactically Immunized with a Live Simian HIV Vaccine Was Dependent on Persistence of the Vaccine Virus

This is a 5-year follow-up study on 12 macaques that were immunized orally with two live SHIV vaccines, six with V1 and six with V2. All 12 macaques became persistently infected after transient replication of the vaccine viruses; all were challenged vaginally 6 mo later with homologous pathogenic SHIVKU-1. Two of the V1 group developed full-blown AIDS without evidence of vaccine virus DNA in tissues. The data on the 10 vaccinated survivors showed that all 10 became infected with SHIVKU-1 and that DNA of both vaccine and SHIVKU-1 viruses were present 6 mo postchallenge, with minimal replication of SHIVKU-1. During the following 5 years, these animals remained persistently infected, but with only one of the two viruses. Six animals eliminated their vaccine virus after variable periods of time and four of these succumbed to reactivation of the challenge virus and AIDS. Five years after challenge, four latently infected animals, two with V2 and two with SHIVKU-1, were reinoculated with SHIVKU-1. This resulted in transient superinfection and the animals promptly returned to their prechallenge status. Immunosuppression of the four animals 1 year later with Abs to CD8+ lymphocytes resulted in transiently productive replication of their respective latent viruses, and upon recovery of CD8+ lymphocytes, they reverted to their latent virus status. The major finding was that of eight animals that eliminated the vaccine virus, six developed AIDS. The two others harboring SHIVKU-1 remain at risk for developing late-onset disease. The primary correlate against AIDS was persistence of the vaccine virus.

Neuropathogenesis of chimeric simian human immunodeficiency virus infection in rhesus macaques.

Comparative studies were performed to determine the neuropathogenesis of infection in macaques with simian human immunodeficiency virus (SHIV)89.6P and SHIVKU. Both viruses utilize the CD4 receptor and CXCR4 co‐receptor. However, in addition, SHIV89.6P uses the CCR5 co‐receptor. Both agents are dual tropic for CD4+ T cells and blood‐derived macrophages of rhesus macaques. Following inoculation into macaques, both caused rapid elimination of CD4+ T cells but they varied greatly in mechanisms of neuropathogenesis. Two animals infected with SHIV89.6P developed typical lentiviral encephalitis in which multinucleated giant cell formation, nodular accumulations of microglial cells, activated macrophages and astrocytes, and perivascular accumulations of mononuclear cells were present in the brain. Many of the macrophages in these lesions contained viral RNA. Three macaques infected with SHIVKU and killed on days 6, 11 and 18, respectively, developed a slowly progressive infection in the CNS but macrophages were not productively infected and there were no pathological changes in the brain. Two other animals infected with this virus and killed several months later showed minimal infection in the brain even though one of the two developed encephalitis of unknown etiology. The basic difference in the mechanisms of neuropathogenesis by the two viruses may be related to co‐receptor usage. SHIV89.6P, in utilizing the CCR5 co‐receptor, caused neuropathogenic effects that are similar to other neurovirulent primate lentiviruses.

Longitudinal study to assess the safety and efficacy of a live-attenuated SHIV vaccine in long term immunized rhesus macaques

Live-attenuated viruses derived from SIV and SHIV have provided the most consistent protection against challenge with pathogenic viruses, but concerns regarding their long-term safety and efficacy have hampered their clinical usefulness. We report a longitudinal study in which we evaluated the long-term safety and efficacy of DeltavpuSHIV(PPC), a live virus vaccine derived from SHIV(PPC). Macaques were administered two inoculations of DeltavpuSHIV(PPC), three years apart, and followed for eight years. None of the five vaccinated macaques developed an AIDS-like disease from the vaccine. At eight years, macaques were challenged with pathogenic SIV and SHIV. None of the four macaques with detectable cellular-mediated immunity prior to challenge had detectable viral RNA in the plasma. This study demonstrates that multiple inoculations of a live vaccine virus can be used safely and can significantly extend the efficacy of the vaccine, as compared to a single inoculation, which is efficacious for approximately three years.